Mycobacterium phlei mutants convert sterols to androsta-1,4-diene-3,17-dione and androsta-4-ene-3,17-dione

ABSTRACT

Mutants which are used in a novel microbiological process to selectively degrade steroids having 17-alkyl side chains of from 2 to 10 carbon atoms, inclusive, to androsta-1,4-diene-3,17-dione (ADD) and androst-4-ene-3,17-dione (AD). ADD and AD are valuable intermediates to make useful steroids.

CROSS REFERENCE TO RELATED APPLICATION

This is a division of application Ser. No. 849,526, filed Nov. 9, 1977,now U.S. Pat. No. 4,293,645, and a continuation-in-part of ourapplication Ser. No. 662,563, filed on Mar. 1, 1976 and now abandoned.

BACKGROUND OF THE INVENTION

The transformation of steroids by microorganisms has been widely studiedand documented. Apparently, the earliest such work was by Mamoli andVercellone in 1937, Ber. 70, 470 and Ber. 70, 2079. They disclosed thereduction of 17-ketosteroids to 17β-hydroxysteroids by fermenting yeast.Since then, Peterson and Murray disclosed the 11α-hydroxylation ofprogesterone with the fungus Rhizopus nigricans; see, U.S. Pat. No.2,602,769 (1952). More recently, Kraychy et al. in U.S. Pat. No.3,684,657 (1972) discloses the selective microbiological degradation ofsteroidal 17-alkyls by fermenting a steroid containing at least 8carbons in the 17-alkyl side chain with Mycobacterium sp. NRRL B-3683 toprepare androst-4-ene-3,17-dione, androst-1,4-diene-3,17-dione, and20α-hydroxymethyl-pregna-1,4-dien-3-one. Even more recently, Marsheck etal. in U.S. Pat. No. 3,759,791 (1973) disclose the selectivemicrobiological preparation of androst-4-ene-3,17-dione by fermenting asteroid of the cholestane or stigmastane series containing at least 8carbons in the 17-alkyl side chain with Mycobacterium sp. NRRL B-3805.

BRIEF SUMMARY OF THE INVENTION

Mutants which are characterized by their ability to selectively degradesteroids having 17-alkyl side chains of from 2 to 10 carbon atoms,inclusive, and accumulate androsta-1,4-diene-3,17-dione, hereinafterreferred to as ADD, and androst-4-ene-3,17-dione, hereinafter referredto as AD, in the fermentation beer. These mutants can be obtained frommicroorganisms of the following genera by using the mutation proceduresdisclosed herein or other mutation procedures: Arthrobacter, Bacillus,Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium,Nocardia, Protaminobacter, Serratia, and Streptomyces. A preferred genusis Mycobacterium. Exemplary species of this genus are M. phlei, M.smegmatis, M. rhodochrous, M. mucosum, M. fortuitum, and M. butyricum.Specifically exemplified herein are the novel mutant microorganisms,Mycobacterium fortuitum, NRRL B-8153, and Mycobacterium phlei, NRRLB-8154, which are used to selectively degrade steroids having 17-alkylchains of from 2 to 10 carbon atoms, inclusive, to ADD and AD. Examplesof suitable steroid substrates are sitosterols, cholesterol,stigmasterol, campesterol, and like steroids with 17-alkyl side chainsof from 2 to 10 carbon atoms, inclusive. These steroid substrates can bein either the pure or crude form.

DETAILED DESCRIPTION OF THE INVENTION The Microorganisms

Mutants which are characterized by their ability to selectively degradesteroids having 17-alkyl side chains of from 2 to 10 carbon atoms,inclusive, and accumulate ADD and AD in the fermentation beer can beobtained by mutating microorganisms of the following genera:Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium,Mycobacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces.Mycobacterium fortuitum, ATCC 6842, and Mycobacterium phlei, UC 3533,have been mutated, as disclosed herein, to give novel laboratory mutantmicrooganisms.

The 1974 ATCC Catalogue discloses the following alongside the listing ofATCC 6842: "J. C. Cruz 2. Cold abscess. Acta Med. Rio de Janeiro 1:1(1936). Medium 90 37C". M. fortuitum, ATCC 6842, degrades sterolsnonselectively to small molecular weight compounds, e.g. CO₂ +H₂ O.Thus, this microorganism is not suitable as a selective steroiddegrader.

Mutation of M. fortuitum, ATCC 6842, and M. phlei, UC 3533 (UC denotesThe Upjohn Company Culture Collection), using nitrosoguanidine hasresulted in the production of novel mutants which selectively degradesteroids having 17-alkyl side chains of from 2 to 10 carbon atoms,inclusive, to produce ADD and AD. These mutant microorganisms have beengiven the accession numbers NRRL B-8153 and NRRL B-8154, respectively,by the Northern Regional Research Laboratory, U.S. Department ofAgriculture, Peoria, Ill., U.S.A., where they have been deposited in thepermanent collection. A subculture of these microorganisms is freelyavailable from this depository by request made thereto. It should beunderstood that the availability of the cultures does not constitute alicense to practice the subject invention in derogation of patent rightsgranted with the subject instrument by governmental action.

The microorganisms of the subject invention have been distinguished fromthe Mycobacterium species NRRL B-3805, disclosed in U.S. Pat. No.3,759,791, which is discussed supra. NRRL B-3805 has the generalcharacteristics of Mycobaterium vaccae which is a distinctly differentspecies than the M. fortuitum and M. phlei of the subject invention. SeeBergey's Manual of Determinative Bacteriology, 8th Edition, The Williamsand Wilkins Company, 1974, on pages 695 and 696 for a comparison ofthese microorganisms.

The morphology and drug sensitivities of M. fortuitum NRRL B-8153 and M.phlei NRRL B-8154, are indistinguishable from that of the parent M.fortuitum, ATCC 6842, and M. phlei, UC 3533, respectively. Both M.fortuitum and M. phlei cultures are acid-fast mon-motile,non-spore-forming bacilli belonging to the family Mycobacteriaceae ofthe order Actinomycetales. According to Runyons classification, Runyon,E. H. 1959 Med. Clin. North America 43:273, M. fortuitum is anonchromogenic group IV mycobacterium, i.e., it grows rapidly at lowtemperatures to produce nonpigmented colonies on relatively simplemedia. M. phlei is also a group IV mycobacterium but produces colonieswhich are deep yellow to orange when grown on simple media.

M. fortuitum ATCC 6842 and M. fortuitum NRRL B-8153, are clearlydistinguishable in their action on steroid molecules. As disclosedabove, M. fortuitum ATCC 6842 is a non-selective degrader of steroids,whereas M. fortuitum NRRL B-8153 is a selective degrader. This propertyof M. fortuitum NRRL B-8153 makes it highly useful, as disclosed herein.Further, M. phlei UC 3533 and M. phlei NRRL B-8154 are alsodistinguishable in their action on steroid molecules. M. phlei UC 3533is a non-selective degrader, whereas NRRL B-8154 is a selectivedegrader.

The mutation of M. fortuitum ATCC 6842 and M. phlei UC 3533 to give M.fortuitum NRRL B-8153 and M. phlei NRRL B-8154, respectively, wasaccomplished by the use of nitrosoguanidine. The details of theprocedure are described infra. Though mutation procedures are generallyknown in the art, there is no known art which teaches or even suggeststhe type of mutants, if any, which might be obtained by use of thesubject mutation procedure. Also, though the mutation and transformationprocedures, disclosed herein, are detailed for a Mycobacterium, itshould be understood that similar or equivalent procedures can be usedwith microorganisms of the other genera, as disclosed herein.

The Transformation Process

The selective transformation of the subject invention can be effected ina growing culture of M. fortuitum NRRL B-8153 or M. phlei NRRL B-8154 byeither adding the selected steroid substrate to the culture during theincubation period, or incorporating it in the nutrient medium prior toinoculation. The steroid can be added singly or in combination withanother steroid. The preferred, but not limiting, range of concentrationof the steroid in the culture is about 0.1 to about 100 grams per liter.The culture is grown in a nutrient medium containing a carbon source,for example, an assimilable carbohydrate, and a nitrogen source, forexample, an assimilable nitrogen compound or proteinaceous material.Preferred carbon sources include glucose, brown sugar, sucrose,glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like.Preferred nitrogen sources include cornsteep liquor, yeast, autolyzedbrewer's yeast with milk solids, soybean meal, cottonseed meal,cornmeal, milk solids, pancreatic digest of casein, fish meal,distillers' solids, animal peptone liquors, meat and bone scraps,ammonium salts and the like. Combinations of these carbon and nitrogensources can be used advantageously. Trace metals, for example, zinc,magnesium, manganese, cobalt, iron, and the like, need not be added tothe fermentation media since tap water and unpurified ingredients areused as components of the medium prior to sterilization of the medium.

The transformation process can range from about 72 hours to 15 days ormore. The incubation temperature can range from about 25° C. to about37° C., with 30° C. being preferred for NRRL B-8153 and 35° C. for NRRLB-8154. The contents are aerated with sterilized air and agitated tofacilitate growth of the microorganism, and, thus, enhance theeffectiveness of the transformation process.

Upon completion of the transformation process, as evidenced by thinlayer chromatography using silica gel plates (E. Merck, Darmstadt) and asolvent system consisting of 2:3 (by volume) ethyl acetate-cyclohexane,the desired transformed steroids are recovered by means well known inthe art. For example, the fermentation (transformation) reactionmixture, including the fermentation liquor and cells, can be extractedwith a water-immiscible organic solvent for steroids. Suitable solventsare dichloromethane (preferred), methylene chloride, chloroform, carbontetrachloride, ethylene chloride, trichloroethylene, ether, amylacetate, benzene and the like.

Alternatively, the fermentation liquor and cells can be first separatedby conventional methods, e.g., filtration or centrifugation, and thenseparately extracted with suitable solvents. The cells can be extractedwith either water-miscible or water-immiscible solvents. Thefermentation liquor, freed of cells, can be extracted withwater-immiscible solvents.

The extracts can be filtered through diatomaceous earth and the filtratevacuum distilled to dryness. The resulting residue containing thedesired transformed steroids then can be dissolved in a minimum of ethylacetate-cyclohexane (20:80). This solution then can be chromatographedon dry silica gel using the solvent system ethyl acetate-benzene(20:80). ADD and AD can be separated from the silica gel by elution withthe solvent system ethyl acetate-chloroform (15:85). The compounds thencan be isolated as separate entities by evaporation of the solvent andrecrystallization from hexane.

The desired products of the subject invention transformation process arethe known steroid intermediates ADD and AD. These compounds are usefulas intermediates in the synthesis of useful steroidal hormones. Forexample, ADD can be used to make estrone according to the processdisclosed in U.S. Pat. No. 3,274,183. Also, AD can be used to maketestosterone according to processes disclosed in U.S. Pat. Nos.2,143,453; 2,253,798; 2,264,888 and 2,356,154.

The following examples are illustrative of the process and products ofthe subject invention but are not to be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Preparation of Mutant M. fortuitum NRRL B-8153 From M.fortuitum ATCC 6842.

(a) Nitrosoquanidine Mutagenesis

Cells of M. fortuitum ATCC 6842 are grown at 28° C. in the followingsterile seed medium:

    ______________________________________                                        Nutrient Broth (Difco) 8 g/liter                                              Yeast Broth            1 g/liter                                              Glycerol               5 g/liter                                              Distilled Water, q.s.  1 liter                                                ______________________________________                                    

The pH is adjusted to 7.0 with 1 N NaOH prior to sterilization at 121°C. for 20 minutes.

The cells are grown to a density of about 5×10⁸ per ml, pelleted bycentrifugation, and then washed with an equal volume of sterile 0.1 Msodium citrate, pH 5.6. Washed cells are resuspended in the same volumeof citrate buffer, a sample removed for titering (cell count), andnitrosoquanidine added to a final concentration of 50 μg/ml. The cellsuspension is incubated at 37° C. in a water bath for 30 minutes, afterwhich a sample is again removed for titering and the remaindercentrifuged down and washed with an equal volume of sterile 0.1 Mpotassium phosphate, pH 7.0. Finally, the cells are resuspended in asterile minimal salts medium, minus a carbon source, consisting of thefollowing:

    ______________________________________                                        NH.sub.4 NO.sub.3    1.0    g/liter                                           K.sub.2 HPO.sub.4    0.25   g/liter                                           MgSO.sub.4.7H.sub.2 O                                                                              0.25   g/liter                                           NaCl                 0.005  g/liter                                           FeSO.sub.4.7H.sub.2 O                                                                              0.001  g/liter                                           Distilled Water, q.s.                                                                              1      liter                                             ______________________________________                                    

The pH is adjusted to 7.0 with 1 N HCl prior to sterilization at 121° C.for 20 minutes. The cells are then plated out to select for mutants.

(b) Selection And Isolation Of Mutant M. fortuitum NRRL B-8153.

Mutagenized cells, as described above, are diluted and spread ontoplates containing a medium consisting of the following (modified fromFraser and Jerrel. 1963. J. Biol. Chem. 205:291-295).

    ______________________________________                                        Glycerol             10.0   g/liter                                           K.sub.2 HPO.sub.4    0.5    g/liter                                           NH.sub.4 Cl          1.0    g/liter                                           MgSO.sub.4.7H.sub.2 O                                                                              0.5    g/liter                                           FeCl.sub.3.6H.sub.2 O                                                                              0.05   g/liter                                           Distilled Water, q.s.                                                                              1      liter                                             ______________________________________                                    

Agar (15 g/liter) is added, and the medium is autoclaved at 121° C. for30 minutes and then poured into sterile Petri plates.

Growth on this medium eliminates most nutritional auxotrophs produced bythe mutagensis procedure, e.g. cultures that require vitamins, growthfactors, etc. in order to grow on chemically defined medium areeliminated. After incubation at 28° C. for about 7 days, the resultingcolonies are replicated to test plates suitable for selecting mutantsand then back onto control plates containing the glycerol-based medium.The test plates are prepared as described by Peterson, G.E., H. L. Lewisand J. R. Davis. 1962. "Preparation of uniform dispersions ofcholesterol and other water-insoluble carbon sources in agar media." J.Lipid Research 3:275-276. The minimal salts medium in these plates is asdescribed above in section (a) of Example 1. Agar (15 g/liter), and anappropriate carbon source (1.0 g/liter), such as sitosterol orandrostenedione (AD), are added and the resulting suspension autoclavedfor 30 minutes at 121° C. The sterile, hot mixture is then poured into asterile blender vessel, blended for several minutes, and then pouredinto sterile Petri dishes. Foaming tends to be a problem in thisprocedure but can be reduced by blending when the mixture is hot and byflaming the surface of the molten agar plates. In this manner uniformdispersions of water-insoluble carbon sources are obtained whichfacilitates the preparation of very homogenous but opaque agar plates.

Colonies which grew on the control plates, but not on test platescontaining AD as the sole carbon source, are purified by streaking ontonutrient agar plates. After growth at 28° C., individual clones arepicked from the nutrient agar plates with sterile toothpicks andretested by inoculating grided plates containing AD as the carbonsource. Purified isolates which still exhibit a phenotype different fromthe parental culture are then evaluated in shake flasks.

(c) Shake Flask Evaluation

Shake flasks (500 ml) contain 100 ml of biotransformation mediumconsisting of the following ingredients:

    ______________________________________                                        NH.sub.4 Cl                                                                             3.0    g/liter                                                      UREA      0.5    g/liter  CaCO.sub.3 3.0 g/liter                              Cerelose  10.0   g/liter  Tween 80*  0.5 g/liter                              KH.sub.2 PO.sub.4                                                                       0.5    g/liter  Tap Water, q.s.                                                                          1   liter                                MgSO.sub.4.7H.sub.2 O                                                                   0.5    g/liter                                                      FeCl.sub.3.6H.sub.2 O                                                                   0.5    g/liter                                                      ______________________________________                                         *Atlas Refinery, Inc., Newark, New Jersey.                               

Soyflour (1 g/liter) is blended into the medium and then sitosterol (30g/liter) is also blended into the medium. After the flasks areautoclaved for 30 minutes at 121° C., they are cooled to 28° C. and theninoculated with 10 ml of seed growth prepared as follows:

The purified isolates from part (b) are grown on agar slants at 28° C. Aloop of cells taken from a slant is used to inoculate a 500-ml flaskcontaining 100 ml of sterile seed medium consisting of the followingingredients:

    ______________________________________                                        Nutrient Broth (Difco) 8 g/liter                                              Yeast Extract          1 g/liter                                              Glycerol               5 g/liter                                              Distilled Water, q.s.  1 liter                                                ______________________________________                                    

The pH is adjusted to 7.0 with 1 N NaOH prior to autoclaving the flasksat 121° C. for 20 minutes. The seed flasks are incubated at 28° C. for72 hours.

As disclosed above, 10 ml of seed growth is then used to inoculate each500-ml flask containing 100 ml of sterile transformation medium. Theflasks are then incubated at 28° C. to 30° C. on a rotary shaker andsampled at various intervals. Ten ml samples are removed and extractedby shaking with 3 volumes of methylene chloride. Portions of theextracts are analyzed by thin layer chromatography using silica gel andthe solvent system described above, i.e., 2:3 (by volume) ethylacetatecyclohexane, and by gas-liquid chromatography. Evidence of thepresence of ADD and AD confirms the selective degradation of sitosterolby the novel mutant produced from the parent M. fortuitum ATCC 6842.

EXAMPLE 2 Transformation of Sitosterol to ADD and AD

The medium used is the same as in Example 1 (c). This medium issterilized by autoclaving 30 minutes at 121° C., whereupon it is cooledto 30° C. and then inoculated with 10 parts of a seed culture of themutant mycobacterium M. fortuitum NRRL B-8153, prepared as described inExample 1(c). The inoculated mixture is incubated at 30° C. for 336hours with agitation to promote submerged growth. Following incubation,the mixture is extracted with dichloromethane. The extract is dried overanhydrous sodium sulfate and the solvent is removed by vacuumdistillation. The resulting residue is dissolved in a minimum of ethylacetate-cyclohexane (20:80). This solution is then chromatographed ondry silica gel using the solvent system ethyl acetate-benzene (20:80).The presence of androst-4-ene-3,17-dione andandrosta-1,4-diene-3,17-dione is shown by thin layer chromatography.These compounds are separated from the silica gel by elution with thesolvent system ethyl acetate-chloroform (15:85). The compounds are thenisolated by evaporation of the solvent and recrystallization fromhexane.

EXAMPLE 3

By substituting M. phlei NRRL B-8154 for M. fortuitum NRRL B-8153 inExample 2, and an incubation temperature of 35° C. for 30° C., also inExample 2, there is obtained a mixture of ADD and AD.

EXAMPLE 4

By substituting cholesterol for sitosterol in Examples 2 and 3 there isobtained a mixture of ADD and AD.

EXAMPLE 5

By substituting stigmasterol in Examples 2 and 3 for sitosterol there isobtained a mixture of ADD and AD.

EXAMPLE 6

By substituting campesterol for sitosterol in Examples 2 and 3 there isobtained a mixture of ADD and AD.

EXAMPLE 7

By adding a combination of any of the steroids in Examples 2-6, inaddition to sitosterol, or in place of sitosterol, in Examples 2 and 3there is obtained a mixture of ADD and AD.

EXAMPLE 8

By substituting a microorganism from the genera Arthrobacter, Bacillus,Brevibacterium, Corynebacterium, Microbacterium, Nocardia,Protaminobacter, Serratia, and Streptomyces, in Example 1 forMycobacterium fortuitum ATCC 6842 and Mycobacterium phlei UC 3533, thereare obtained mutant/microorganisms which are characterized by theirability to selectively degrade steroids having 17-alkyl side chains offrom 2 to 10 carbon atoms, inclusive, and accumulate ADD and AD in thefermentation beer.

EXAMPLE 9

By substituting the mutants obtained in Example 8 for M. fortuitum NRRLB-8153 and M. phlei NRRL B-8154 in Examples 2-7, there are obtained ADDand AD.

We claim:
 1. A process for preparing a mixture ofandrosta-1,4-diene-3,17-dione and androst-4-ene-3,17-dione whichcomprises cultivating Mycobacterium phlei NRRL B-8154 in an aqueousnutrient medium under aerobic conditions in the presence of a steroidcontaining from 2 to 10 carbon atoms, inclusive, in the 17-alkyl sidechain and recovering androsta-1,4-diene-3,17-dione andandrost-4-ene-3,17-dione.
 2. A process, according to claim 1, whereinsaid steroid is sitosterol.
 3. A process, according to claim 1, whereinsaid steroid is cholesterol.
 4. A process, according to claim 1, whereinsaid steroid is stigmasterol.
 5. A process, according to claim 1,wherein said steroid is campesterol.
 6. A process for preparing amixture of androsta-1,4-diene-3,17-dione and androst-4-ene-3,17-dionewhich comprises cultivating Mycobacterium phlei NRRL B-8154 in anaqueous nutrient medium under aerobic conditions in the presence of amixture of two or more steroids wherein each steroid contains from 2 to10 carbon atoms, inclusive, in the 17-alkyl chain and recoveringandrosta-1,4-diene-3,17-dione and androst-4-ene-3,17-dione.
 7. Aprocess, according to claim 6, wherein said steroid mixture is selectedfrom the group consisting of sitosterol, cholesterol, stigmasterol, andcampesterol.
 8. A process for preparing a mixture ofandrosta-1,4-diene-3,17-dione and androst-4-ene-3,17-dione whichconsists of cultivating a mutant microorganism of Mycobacterium phlei,said mutant being characterized by its ability to selectively degradesteroids having 17-alkyl side chains of from 2 to 10 carbon atoms,inclusive, and accumulate androsta-1,4-diene-3,17-dione andandrost-4-ene-3,17-dione in the fermentation beer, in an aqueousnutrient medium under aerobic conditions in the presence of a steroidcontaining from 2 to 10 carbon atoms, inclusive, in the 17-alkyl sidechain and recovering androsta-1,4-diene-3,17-dione andandrost-4-ene-3,17-dione.